Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB

Simplistic Attachment and Multispectral Imaging with Semiconductor Nanocrystals

Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC’s) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC’s with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry. Here we report the development of simple, yet novel attachment chemistries for antibodies onto NC’s and demonstrate how spectral deconvolution technology enables the multiplexed detection of 5 distinct NC-antibody conjugates with fluorescence emission wavelengths separated by as little as 20 nm

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Photothermal Ablation Of Amyloid Aggregates By Gold Nanoparticles

Amyloid peptide (Aβ) is found in the brain and blood of both healthy and diseased individuals alike. However, upon secondary structure transformation to a β-sheet dominated conformation, the protein aggregates. These aggregates accumulate to form neuritic plaques that are implicated in the pathogenesis of Alzheimer\u27s disease. Gold nanoparticles are excellent photon-thermal energy converters. The extinction coefficient of the surface plasmon band of gold nanoparticles is very large when compared to typical organic dyes. In this study, gold nanoparticle-Aβ conjugates were prepared and the photothermal ablation of amyloid peptide aggregates by laser irradiation was studied. Monofunctional gold nanoparticles were prepared using a recently reported solid phase modification method and then coupled to fragments of Aβ peptide, namely Aβ(31-35) and Aβ(25-35). The conjugates were then mixed with Aβ fragments in solution. The aggregated peptide formation was studied by a series of spectroscopic and microscopic techniques. The peptide aggregates were then irradiated by a continuous laser. With gold nanoparticle-Aβ conjugates present the aggregates were destroyed by photothermal ablation. Gold nanoparticles without Aβ conjugation were not incorporated into the aggregates and when irradiated did not result in photothermal ablation. With gold nanoparticle-Aβ conjugates the ablation was selective to the site of irradiation and minimal damage was observed as a result of thermal diffusion. In addition to the application of photoablation to a protein-based sample the nanoparticles and the chemistry involved provide an easily monofunctionalized photothermal material for the biological conjugation. © 2008 Elsevier B.V. All rights reserved

A Two-Dimensional Approach to Study Amyloid β-Peptide Fragment (25−35)

The neuritic plaques formed by amyloid β-peptide (Aβ) play a seminal role in the pathogenesis of Alzheimer's disease (AD). Aβ sequence 25−35 (GSNKGAIIGLM) is among the most frequently studied Aβ derivatives for the reason that it possesses the structural characteristic of Aβ and remains neurotoxic. Aβ(25−35) was modified with an aliphatic chain (C18) at the N-terminal of the peptide for the study of the Langmuir monolayer at the air−water interface. The main advantage of the 2D approach is the self-assembly of the peptide moiety in the subphase, and therefore the aggregation process of the peptidolipid Aβ(25−35) was monitored by surface pressure and surface potential−area isotherms. The real-time epifluorescence microscopy was utilized to observe the topography of the domains formed, whereas polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) provided the information on the structural features of the domains at the air−water interface. Langmuir−Blodgett films were prepared to examine by circular dichroism (CD) the conformation of the peptidolipid film in the domains

Antibody-gold quantum dot-PAMAM dendrimer complex as an immunoglobulin immunoassay

Gold quantum dots (AuQDs) were synthesized and electrostatically conjugated to goat-derived anti-human IgG for the purpose of detecting human IgG in solution over a broad range of concentrations. The system is able to detect human IgG by linear fluorescence quenching over a micromolar to nanomolar concentration range. We have demonstrated the specificity and a wide dynamic range of the proposed immunoassay. The quenching is a result of competitive surface quenching of the AuQDs. Characterization, details of the immunoassay, and the quenching mechanism, are discussed

Immunoasssay based on the antibody-conjugated PAMAM-dendrimer-gold quantum dot complex

An immunoassay based upon photoluminescent gold quantum dots aimed at detecting human IgG in aqueous solution from micromolar to nanomolar concentrations is described